LOCUS       sequence-486014-        5148 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  attP Hyg Resistant plasmid for RMI targeting an attB site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5148)
  AUTHORS   Nonet ML
  TITLE     Creation and manipulation of bipartite expression transgenes in C. 
            elegans using phiC31 recombinase.
  JOURNAL   bioRxiv 2024.03.01.583017
REFERENCE   2  (bases 1 to 5148)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.03.01.583017"
COMMENT     Sequence Label: pAttPF3Hyg
FEATURES             Location/Qualifiers
     source          1..5148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    734..767
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1142..1166
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1167..2192)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1167..2192)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     protein_bind    complement(2195..2219)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    2618..2651
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     2672..2689
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2805..2826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     2882..2899
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2883..2899
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2909..2928
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2909..2927
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3076..3096)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3076..3094)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3115..3131)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3115..3131)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3128..3150)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3139..3155
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(3380..3399)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3590..3611
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    3658..3757
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
     promoter        3971..4075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4076..4144,4145..4936)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4294..4313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5107..5148,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaatg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gaagttccta ttcttcaaat agtataggaa cttcaccggg
      721 ctgcagaggc cgtataactt cgtataaagt atcctatacg aagttatccc agttgtcctc
      781 gaacaggtct aaaaattgcg gtcataaact gaaacgtaac atatgataag gtattttgtg
      841 tgcggttttt tctatgatgt ttcaaaaaaa tttatcagaa gtaaaaaaca taagaagtgt
      901 acacagaaca ttgtgtagat tttgggatag aattattatt aatgggggga gggagcacaa
      961 tttttttcga ctttttatta cgaattaatt ctatttttga atctacacaa tttcattgtt
     1021 agaggtgact taaaagaagc taaaaaacaa agaaattaag agaagttttt tgataataac
     1081 aaaaataggg ggtgggagca cagggagaaa gagcatgtag ggatgttgaa gagtaattgg
     1141 acccactttg tacaagaaag ctgggtctat tcctttgccc tcggacgagt gctggggcgt
     1201 cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg
     1261 cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga
     1321 ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca
     1381 agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc
     1441 cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg
     1501 ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt
     1561 atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg
     1621 acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca
     1681 ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat
     1741 atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg
     1801 ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggctgcaga
     1861 acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag
     1921 atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc
     1981 gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta
     2041 tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg
     2101 ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg
     2161 acagacgtcg cggtgagttc aggctttttc atggagcctg cttttttgta caaacttgcg
     2221 gcaccgcctg acatattacc ttaaaattca aaaattaatt tcagatcagt cataaccaag
     2281 aaaacaaaat aaacgttagg acagaaaatt caaattaatt aaaaattatg ctgaataaga
     2341 aaattacatg caaagcctta ttttcctcat agaaacagtt gaataacata ttttttaatt
     2401 gaaaacatag caaaattcgt ggtaaaattt gtcaaaacct tggaaaaaat cgaaaaaaga
     2461 acatttcccg atgcattttc ctatttctcg ttgccgacaa cgtataaacg ttgaatactc
     2521 agtcaacgaa tttggctgct ggcttggact ctcgcacgga agagacgcgt gtgtagattt
     2581 acgacgaaag caaaaatgat cccgctttcc agtcgggata acttcgtata aagtatccta
     2641 tacgaagtta tgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaatt
     2701 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2761 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     2821 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgcc agtcacgacg
     2881 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     2941 cgggcccccg gttaacgggt cgacggtatc gataagctta ccgaagagca ggcctgcaca
     3001 tatgttacta gtggatccat gggctcttcg ccatctagag cggccgccac cgcggtggag
     3061 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     3121 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaagaaaa ccctggcgtt
     3181 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     3241 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     3301 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3361 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3421 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3481 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3541 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3601 ttccaaactg gaacaacact caacgtaagc acccgcgtac gtgtccaccc cggtcacaac
     3661 cccttgtgtc atgtcggcga ccctacgccc ccaactgaga gaactcaaag gttaccccag
     3721 ttggggcact actcccgaaa accgcttctg acctgggaaa acgtgaagcc ccggggcatc
     3781 cgctgagggt tgccgccggg gcttcggtgt gtccgtcagt acctatctcg gtctattctt
     3841 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3901 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3961 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4021 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4081 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4141 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4201 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4261 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4321 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4381 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4441 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4501 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4561 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4621 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4681 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4741 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gatctcgcgg
     4801 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4861 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4921 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4981 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5041 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5101 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaac
//
